The present invention relates to peptides, which enhance uptake of a pharmaceutically active agent into a cell, into or out of an intracellular compartment, and across a cell layer. More particularly, the present invention relates to membrane translocating peptides, fragments, motifs, derivatives, analogs or peptidomimetics thereof and to the nucleotide sequences coding therefor, which enhance uptake of a pharmaceutically active agent into a cell, into or out of an intracellular compartment, and across a cell layer either directly or from a pharmaceutically active agent loaded particle.
The epithelium lining the gastrointestinal tract (hereinafter, xe2x80x9cGITxe2x80x9d) is a major barrier to absorption of orally administered pharmaceutically active agents (hereinafter, xe2x80x9cactive agentsxe2x80x9d). Absorption across the GIT epithelium can be transcellular transport through the cells and by paracellular transport between the cells. Transcellular transport includes, but is not limited to, receptor-mediated, transporter-mediated, channel-mediated, pinocytotic and endocytotic mechanisms and to diffusion. Paracellular transport includes, but is not limited to, movement through right junctions. Of particular interest is the development of non-invasive methods for enhancing uptake of active agents across the GIT epithelium into the body (Evers, P. Developments in Drug Delivery: Technology and Markets, Financial Times Management Report, 1995).
To develop non-invasive methods, phage display libraries have been used to identify specific peptide sequences, which bind preferentially to specific GIT membrane receptor, transporter, channel, pinocytotic or endocytotic target pathways (hereinafter, xe2x80x9ctargeting peptidesxe2x80x9d) within the GIT. Included among the target pathways, which have been screened with phage display libraries, are the GIT membrane transporters HPT1, hPEPT1, D2H and hSI. HPT1 and hPEPT1 transport dipeptides and tripeptides. D2H transports neutral and basic amino acids and is a transport activating protein for a range of amino acid translocases. hSI is involved in sugar metabolism and comprises 9% of the brush border protein in the jejunum. Specific peptide sequences, which interact with the HPT1, hPEPT1, D2H and hSI membrane transporters have been identified in U.S. patent application Nos. 09/079,819, 09/079,723 and 09/079,678 (hereby incorporated by reference in their entireties).
Non-target pathway based assays have been used to identify peptides with inherent cell membrane translocating properties. These cell membrane translocating peptides interact directly with and penetrate the lipids of cell membranes (Fong et al. Drug Development Research 33:64, 1994). The central hydrophobic h-region of the signal sequence of Kaposi""s fibroblast growth factor, AAVLLPVLLAAP (SEQ ID NO: 1) is considered to be a membrane translocating peptide. This peptide (SEQ ID NO: 1) has been used as a carrier to deliver various short peptides ( less than 25 mer), through the lipid bilayer, into living cells in order to study intracellular protein functions and intracellular processes (Lin et al., J. Biol. Chem. 271:5305, 1996; Liu et al. Proc. Natl. Acad. Sci. USA 93:11819, 1996; Rojas et al. J. Biol. Chem. 271:27456, 1996; Rojas et al. Biochem. Biophys. Res. Commun. 234:675, 1997). A 41-kDa glutathione S-transferase fusion protein containing SEQ ID NO:1 (GST-Grbs-SH2 fused to SEQ ID NO: 1) has been shown to be imported into NIH 3T3 fibroblasts and to inhibit epidermal growth factor induced EGFR-Grb2 association and MAP kinase activation (Rojas et al. Nature Biotechnology 16:370, 1998). However, these studies do not address the use of membrane translocating peptides to enhance active agent uptake into a cell, into and out of an intracellular compartment, or across a cell layer when the active agent is complexed to a membrane translocating peptide or when the active agent is incorporated into a particle and the particle is modified with (hereinafter, xe2x80x9ccomplexed toxe2x80x9d) a membrane translocating peptide.
The ability to enhance movement of an active agent across a cell membrane is important because, although an active agent can be administered to an animal by a variety of routes including, but not limited to, oral, nasal, mucosal topical transdermal, intravenous, intramuscular, intraperitoneal, intrathecal and subcutaneous, oral administration is the preferred route. Nasal, mucosal, topical and transdermal administration depend on drug absorption through the mucosa or skin into the circulation. Intravenous administration can result in adverse effects from rapid accumulation of high concentrations of drug, in patient discomfort and in infection at the injection site. Intramuscular administration can cause pain at the injection site. Subcutaneous administration is not suitable for large volumes or for irritating substances. Although oral administration is the preferred route, many active agents are not absorbed efficiently across the GIT epithelium. This results from enzymatic degradation of active agents within the human lumen of the GIT, from the limited permeability of the GIT epithelium to active agents, from the large molecular size of active agents and from the hydrophilic properties of active agents (Fix, J. A. J. Pharmac. Sci. 85:1282, 1996). To develop an oral formation, an active agent must be protected from enzymatic digestion within the lumen of the GIT, presented to the absorptive epithelial cells of the GIT in an effective concentration and xe2x80x9cmovedxe2x80x9d across the epithelium in an apical to basolateral direction.
Therefore, because of the advantages of oral drug administration, there is a need for delivery systems, which protect orally ingested active agents from enzymatic degradation within the lumen of the GIT and which promote the absorption of orally ingested active agents into and across the epithelial cells lining the GIT.
The present invention fulfills this need by providing a membrane translocating peptide comprising a full-length peptide, derivative, fragment, motif, analog or peptidomimetic thereof (hereinafter, xe2x80x9cMTLPxe2x80x9d) or nucleotide sequences coding therefore, a MTLP-active agent complex and a MTLP-active particle complex, wherein the MTLP enhances movement of the active agent or the active particle across a lipid membrane. More particularly, the present invention provides a MTLP, a MTLP-active agent complex and a MTLP-active particle complex, wherein the MTLP enhances movement of the active agent or of the active particle into a cell, into and out of an intracellular compartment and across a cell layer in an animal, including a human. Methods of making and methods of using MTLPs, MTLP-active agent complexes and MTLP-active particle complexes also are included.
MTLPs of the present invention are capable of displaying one or more known functional activities associated with a full-length MTLP. Such functional activities include, but are not limited to, the ability to interact with a membrane and the ability to compete for transport of a reporter drug molecule (fMLP) across epithelial cells including, but not limited to, polarized, differentiated human derived Caco-2 cells. Additional functional activities include, but are not limited to, antigenicity, which includes, but is not limited to, the ability to bind an anti-MTLP antibody and the ability to compete with a MTLP for interaction with a membrane; and, immunogenicity, which includes, but is not limited to, the ability to stimulate antibody generation.
Methods of making a MTLP-active agent complex include, but are not limited to, covalent coupling of a MTLP and an active agent and noncovalent coupling of a MTLP and an active agent. Methods of making a MTLP-active particle complex include, but are not limited to, incorporating an active agent into a particle including, but not limited to, a nanoparticle, a microparticle, a capsule, a liposome, a non-viral vector system and a viral vector system. The MTLP can be complexed to the active particle by methods including, but not limited to, adsorption to the active particle, noncovalent coupling to the active particle and covalent coupling, either directly or via a linker, to the active particle, to the polymer or polymers used to synthesize the active particle, to the monomer or monomers used to synthesize the polymer, and to other components comprising the active particle.
The present invention also includes the nucleotide sequences, which code for the MTLPs. Methods of making nucleotide sequences include, but are not limited to, recombinant means.
MTLPs, MTLP-active agent complexes and MTLP-active particle complexes can be used alone, in combination with or conjugated to other molecules including, but not limited to, molecules that bind to target pathways, to nuclear uptake pathways and to endosomal pathways, molecules that enable mucoadhesion, molecules that facilitate diffusion across lipid membranes or through water filled pores and molecules that regulate or direct intra-cellular trafficking. That is, by using different mechanisms simultaneously, active agent bioavailability may be enhanced.
Therefore it is an object of the present invention to provide a full-length MTLP.
Another object of the present invention is to provide fragments, motifs, derivatives, analogs and peptidomimetics of a full-length MTLP.
Another object of the present invention is to provide a composition comprising an MTLP-active agent complex.
Another object of the present invention is to provide a composition comprising an MTLP-active particle complex.
Another object of the present invention is to provide a composition comprising an MTLP-active particle complex, wherein the particle is a microparticle.
Another object of the present invention is to provide a composition comprising an MTLP-active particle complex, wherein the particle is a nanoparticle.
Another object of the present invention is to provide a composition comprising an MTLP-active particle complex, wherein the particle is a liposome.
Another object of the present invention is to provide a composition comprising a viral DNA particle, wherein the viral particle is modified to express a MTLP on its surface.
Another object of the present invention is to provide a composition comprising a viral DNA particle, wherein the viral particle is complexed to a MLTP following virus production and purification.
Another object of the present invention is to provide a composition comprising a viral DNA particle, wherein the viral particle is complexed to a MTLP following virus production in and purification from a mammalian cell.
Another object of the present invention is to provide a composition comprising a non-viral based gene delivery system, wherein the non-viral based gene delivery system is complexed to a MTLP.
Another object of the present invention is to enhance the movement of an active agent across a lipid membrane.
Another object of the present invention is to enhance the uptake of an active agent into a cell.
Another object of the present invention is to enhance the uptake of an active agent across a cell layer.
Another object of the present invention is to enhance the uptake of an active agent into an epithelial cell.
Another object of the present invention is to enhance the uptake of an active agent across an epithelial cell layer.
Another object of the present invention is to enhance the uptake of an active agent across the epithelial cell layer lining the GIT into the circulation of an animal.
Another object of the present invention is to enhance the movement of an active particle across a lipid membrane.
Another object of the present invention is to enhance the uptake of an active particle into a cell.
Another object of the present invention is to enhance the uptake of an active particle across a cell layer.
Another object of the present invention is to enhance the uptake of an active particle into an epithelial cell.
Another object of the present invention is to enhance the uptake of an active particle across an epithelial cell layer.
Another object of the present invention is to enhance the uptake of an active particle across the epithelial cell layer the GIT into the circulation of an animal.
Another object of the present invention is to provide intracellular gene delivery by a non-viral based gene delivery system.
Another object of the present invention is to provide intracellular gene delivery by a non-viral based gene delivery system, wherein the non-viral based gene delivery system is complexed to a MTLP.
Another object of the present invention is to provide a rapid screening method to identify MTLPs, which retain the essential functional activity of the full-length MTLP.
Another object of the present invention is to provide cell-based screens for assaying the functional activity of a MTLP.
Another object of the present invention is to provide cell-based screens for characterizing the properties of a MTLP.
Another object of the present invention is to provide a method for diagnosing a pathological disorder by oral administration of an amount of a MTLP-active agent complex, wherein the active agent is a diagnostic agent, such that the systemic concentration of the diagnostic agent is effective to diagnose the pathological disorder.
Another object of the present invention is to provide a method for preventing a pathological disorder by oral administration of a MTLP-active agent complex, wherein the active agent is a prophylactic agent, such that the systemic concentration of the prophylactic agent is effective to prevent the pathological disorder.
Another object of the present invention is to provide a method for treating a pathological disorder by oral administration of a MTLP-active agent complex, wherein the active agent is a therapeutic agent, such that the systematic concentration of the therapeutic agent is effective to treat the pathological disorder.
Another object of the present invention is to provide a method for diagnosing a pathological disorder by oral administration of a MTLP-active particle complex, wherein the active particle contains a diagnostic agent, such that the systematic concentration of the diagnostic agent is effective to diagnose the pathological disorder.
Another object of the present invention is to provide a method for preventing a pathological disorder by oral administration of a MTLP-active particle complex, wherein the active particle contains a prophylactic agent, such that the systemic concentration of the prophylactic agent is effective to prevent the pathological disorder.
Another object of the present invention is to provide a method for treating a pathological disorder by oral administration of a MTLP-active particle complex, wherein the active particle contains a therapeutic agent such that the systemic concentration of the therapeutic agent is effective to treat the pathological disorder.
Other objectives, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.